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Thierry Gefflaut

Mélanie Hall

Tamara Reiter

Mélanie Bordeaux

Nicolas Brun

Jullien Drone

Innovative Carrier Materials for 
Advancing Enzyme Immobilization in 
Industrial Biocatalysis. 
 
Quentin Hanniet,1,2 Coline Mateos,1 Ludivine Onillon,1 Awilda Maccow,1 Thierry Gefflaut,3 
Melanie Hall,4 Tamara Reiter,4 Mélanie Bordeaux,5 Nicolas Brun,1,2 Jullien Drone*1,2  
 
1 ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France 
2 CarboZym SAS, Cap Alpha, 3 avenue de l’Europe - 34830 Clapiers, France 
3 Université Clermont Auvergne, Clermont-Auvergne INP, CNRS, Institut de Chimie de 
Clermont-Ferrand, 63000 Clermont-Ferrand, France 
4 Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria. 
5 Fundacion Nicafrance, Finca La Cumplida, Matagalpa, Nicaragua. 
Supporting information 
 
 
Supporting information 1 
Additional figures 3 
Experimental section 4 
Cryogel preparation: 4 
Case studies: 4 
CalB experimentation: 4 
Enzyme Immobilization 4 
Enzyme Quantification by Residual Activity 4 
Activity of Immobilized Enzyme 4 
Continuous Use of CalB Enzyme 4 
Enzymatic Activity in Continuous Flow 4 
GsOYE experimentation: 5 
Enzyme Immobilization (Batch Mode) 5 
2 
 
Enzyme Quantification by Bradford Assay 5 
Enzyme Activity of Immobilized GsOYE 5 
Continuous Use of GsOYE Enzyme 5 
B9L0N2 experimentation: 6 
Transformation (Under Sterile Conditions) 6 
Plasmid Preparation 6 
Plasmid Dilution and Transformation 6 
Heat Shock 6 
Plating 6 
Culture (Under Sterile Conditions) 6 
Pre-culture 6 
Main Culture 6 
Induction 6 
Purification 7 
Cell Lysis 7 
Centrifugation 7 
Affinity Chromatography 7 
Enzyme Immobilization (Batch Mode) 8 
Enzyme Quantification by Bradford Assay 8 
Enzyme Activity of Immobilized B9L0N2 8 
Continuous Use of B9L0N2 Enzyme 8 
 
 
3 
 
Additional figures 
 
Figure S1-a) XPh CarboZym support pictures at macroscopic & microscopic (SEM & TEM) level; b) Adsorption-
desorption isotherms of nitrogen on XPh & XCat materials; c) BJH pore distribution from XPh & XCat materials 
 
 
Figure S2- Continuous flow setups a) HPLC pump (Jasco PU-4180) b) thermostatted column c) Circulation mode d) 
Recirculation mode 
 
4 
 
Experimental section 
Cryogel preparation: 
Gels  were synthesized using xylose and phloroglucinol or catechin according to protocol described in 
patents WO2024153593A1 & WO2024153594A1. 
Case studies: 
CalB experimentation: 
Enzyme Immobilization 
25 mg of cryogel were weighed and added to 1 mL of a Tris buffer solution (20 mM, pH 7) containing 
2.5 mg of desalted commercial CalB enzyme. The suspension was stirred at 800 rpm for 2 hours at 4°C. 
Samples were taken at 10, 20, 30, 60, 90, and 120 minutes for kinetic monitoring. After each sampling, 
the suspension was centrifuged at 4000–6000 rpm for 2–5 minutes. After the final sample, the 
supernatant was discarded, and the solid was washed twice with 1 mL of Tris buffer, with each wash 
involving stirring at 4°C for 3 minutes followed by centrifugation. 
Enzyme Quantification by Residual Activity 
The enzyme content in each supernatant sample was quantified through residual activity assays. For 
each sample, 20 µL of the supernatant was added to a reaction mixture containing 1.5 mM p-
nitrophenyl butyrate (pNPB), 1% (wt) Genapol, and 20 mM Tris-HCl buffer (pH 7). The mixture was 
stirred for 5 minutes at 25°C, and the formation of p-nitrophenol was monitored at 410 nm. The 
resulting absorbance values were compared to a standard curve to determine the protein 
concentration in the supernatant, which was then used to calculate the amount of protein leached 
from the cryogel during immobilization. 
Activity of Immobilized Enzyme 
The hydrolytic activity of the immobilized CalB was assessed by measuring the conversion of pNPB to 
p-nitrophenol. The reaction was carried out in a 1 mL solution containing 15 mM pNPB in 20 mM Tris-
HCl (pH 7) with 1% (wt) Genapol. The reaction was initiated by adding 25 mg of the enzyme-loaded 
cryogel (10% wt enzyme). The mixture was stirred at 800 rpm for 1 hour at 25°C. Enzyme activity was 
determined over five reaction cycles, with each cycle involving the same procedure followed by 
centrifugation, washing with buffer, and re-initiation of the reaction. 
Continuous Use of CalB Enzyme 
In continuous operation (see figure S2 for continuous flow set-up), approximately 65 mg of cryogel, 
pre-loaded with immobilized CalB, was placed in a thermostatted glass column maintained at 4°C using 
a cryostat. The enzyme solution, prepared in Tris buffer (20 mM, pH 7), was percolated through the 
column at a flow rate of 0.1 mL/min using an HPLC pump. Following enzyme immobilization, the 
column was flushed with 5 mL of Tris buffer at the same flow rate to remove unbound proteins. 
Enzymatic Activity in Continuous Flow 
The continuous enzymatic reaction was performed by circulating a reaction mixture containing 5 mM 
p-nitrophenyl butyrate (pNPB) in Tris-HCl buffer (20 mM, pH 7) with 1% (wt) Genapol through the 
column. In recirculation mode, 13.3 mL of the reaction mixture was cycled through the column at 0.5 
mL/min, with 100 µL samples taken hourly. The formation of p-nitrophenol was monitored at 410 nm, 
5 
 
and the enzyme activity was calculated based on the quantity of p-nitrophenol produced, normalized 
to the mass of immobilized enzyme. 
GsOYE experimentation: 
Enzyme Immobilization (Batch Mode) 
In batch mode, 10 mg of cryogel was weighed and added to a 1 mL solution of Tris-HCl buffer (50 mM 
NaCl, pH 7.5) containing 1 mg of GsOYE enzyme, resulting in a 10% enzyme loading on the cryogel. The 
suspension was stirred at 800 rpm for 2 hours at 4°C to facilitate enzyme immobilization. Samples were 
taken at intervals (10, 20, 30, 60, 90, and 120 minutes), followed by centrifugation at 4000–6000 rpm 
for 2 to 5 minutes to separate the solid cryogel from the supernatant. After the final sampling, the 
supernatant was discarded, and the solid cryogel was washed twice with the buffer solution to remove 
unbound enzyme. Each wash involved stirring the suspension for 3 minutes at 4°C, followed by 
centrifugation. 
Enzyme Quantification by Bradford Assay 
The amount of enzyme immobilized on the cryogel was determined using the Bradford protein assay. 
A standard curve was prepared using BSA (0.1 g/L) in the same Tris-HCl buffer (50 mM NaCl, pH 7.5). 
Bradford reagent was added to each sample, including the immobilized enzyme supernatant samples, 
and incubated for 15 minutes in the dark. The absorbance was measured at 595 nm. The protein 
content in the samples was calculated by comparison with the standard curve, and the amount of 
enzyme immobilized was determined based on the amount of protein remaining in the supernatant 
after immobilization. 
Enzyme Activity of Immobilized GsOYE 
The catalytic activity of immobilized GsOYE was measured by following the reduction of cyclohexenone 
to cyclohexanone. The reaction mixture contained 50 mM cyclohexenone, 75 mM NADH, and 2% 
DMSO in Tris-HCl buffer (50 mM NaCl, pH 7.5). The reaction was initiated by adding 10 mg of enzyme-
loaded cryogel to the mixture and stirred at 30°C for 60 minutes at 800 rpm. NADH consumption was 
monitored at 340 nm using UV spectrophotometry, and enzyme activity was calculated based on the 
amount of NADH consumed. The activity of the immobilized enzyme was evaluated over five reaction 
cycles, with each cycle involving the same process followed by washing the cryogel and re-initiating 
the next reaction. 
Continuous Use of GsOYE Enzyme 
In continuous mode (see figure S2 for continuous flow set-up), 50 mg of cryogel was placed in a 
thermostatted glass column maintained at 4°C using a cryostat. The enzyme solution, prepared in Tris-
HCl buffer (50 mM NaCl, pH 7.5), was percolated through the column at a flow rate of 0.1 mL/min using 
an HPLC pump in order to result in a 10% enzyme loading. Following enzyme immobilization, the 
column was flushed with 5 mL of Tris buffer at the same flow rate to remove unbound proteins. The 
continuous enzymatic reaction was performed by circulating a reaction mixture containing 50 mM 1-
cylclohexenone in 200m mM Tris-HCl-NaCl buffer (50 mM NaCl, 2% DMSO, pH 7.5) through the column. 
In recirculation mode, 6 mL of the reaction mixture was cycled through the column at 1 mL/min for up 
to 5 hours, with 100 µL samples taken hourly. The consumption of NADH was monitored at 340 nm to 
determine conversion ratio. 
6 
 
B9L0N2 experimentation: 
Transformation (Under Sterile Conditions) 
Plasmid Preparation 
Dehydrated plasmids were re-suspended in 5 mM Tris-HCl buffer, pH 8.5, as recommended by 
TwistBioscience (the plasmid supplier), to achieve a final concentration of 10 ng/µL. The suspension 
was gently mixed by tapping and incubated at 30°C for 15 minutes without agitation. 
Simultaneously, BL21(DE3) and DH5α competent cells were thawed on ice. 
Plasmid Dilution and Transformation 
The plasmid suspension was further diluted 1:10 with the same buffer to obtain a final DNA 
concentration of 1 ng/µL. Subsequently, 5 µL of this diluted plasmid solution was added to 50 µL of 
competent cells. The mixture was gently mixed by tapping and incubated on ice for 30 minutes. 
Heat Shock 
For heat shock transformation, the following conditions were applied: 
• BL21(DE3) cells: Incubated at 42°C for 30 seconds followed by 2 minutes on ice. 
• DH5α cells: Incubated at 42°C for 20 seconds followed by 2 minutes on ice. 
After heat shock, SOC medium was added: 950 µL for DH5α cells and 250 µL for BL21(DE3) cells. The 
suspensions were incubated at 37°C for 1 hour at 225 rpm. In parallel, pre-poured LB agar plates 
containing 50 µg/mL kanamycin were removed from cold storage and placed in the incubator to 
equilibrate to room temperature, avoiding thermal shock. 
Plating 
The transformed cells were then plated on the LB agar kanamycin plates and incubated overnight at 
37°C. 
Culture (Under Sterile Conditions) 
Pre-culture 
For pre-culture, three isolated colonies from the transformation plates were selected and inoculated 
into a Falcon tube containing 5 mL of LB medium supplemented with 50 µg/mL kanamycin. The cultures 
were incubated overnight at 37°C with shaking at 180 rpm. After incubation, 800 µL of the pre-culture 
was mixed with 800 µL of sterile 50% glycerol to prepare glycerol stocks, which were stored at -80°C 
(N2A03 room). 
Main Culture 
For the main culture, 200 mL of LB medium with 50 µg/mL kanamycin in a non-baffled 1L Erlenmeyer 
flask was inoculated with 2 mL of pre-culture and incubated at 37°C with shaking at 180 rpm until the 
optical density at 600 nm (OD600) reached between 0.4 and 0.6 (approximately 3 hours). 
Induction 
To induce protein expression, 200 µL of 1 M IPTG was added to the culture, yielding a final IPTG 
concentration of 1 mM. The flask was then incubated at 25°C with shaking at 180 rpm for 5 hours. 
7 
 
Purification 
Cell Lysis 
The cell pellet from 100 mL of culture was re-suspended in lysis buffer (see buffer composition table 
below) to achieve a suspension with an OD of 50. Cells were lysed using sonication with a 10 mm probe 
at 40% amplitude for 10 minutes, alternating between 15 seconds ON and 15 seconds OFF. The 
samples were kept on ice throughout the sonication process to prevent overheating and protein 
degradation. 
Centrifugation 
The lysate was centrifuged at 14,000 rpm for 30 minutes at 4°C, and 20 µL of the supernatant was 
collected for further analysis. 
Affinity Chromatography 
The supernatant was loaded onto a 1 mL HisTrap column using the ÄKTA system with a flow rate of 1 
mL/min. The following elution steps described in table S1: 
Step Solution Volume (mL) Fraction Recovered 
Ni Recharging 0.1 M NiSO₄ solution 5 / 
Equilibration Lysis buffer 15 / 
Sample Loading Supernatant X Unbound fraction X mL 
Washing Wash buffer 15 Wash fraction 15 mL 
Elution Elution buffer 10 Eluted fraction 10 x 1 mL 
Table S1: Elution steps and corresponding solution 
The most concentrated elution fractions were pooled and subjected to desalting using a 5 mL Desalting 
column on the ÄKTA system at a flow rate of 3 mL/min. The desalting steps are described in table S2 
& S3: 
Step Solution Volume (mL) Fraction Recovered 
Equilibration Desalting buffer 100 / 
Sample Loading Eluted fractions X / 
Elution Desalting buffer 10 Desalted fraction 10 x 1 mL 
Table S2: Desalting steps and corresponding solution 
 
Buffer Composition 
Lysis buffer 50 mM KPi, 300 mM NaCl, 10 mM Imidazole, pH 8 
Wash buffer 50 mM KPi, 300 mM NaCl, 20 mM Imidazole, pH 8 
Elution buffer 50 mM KPi, 300 mM NaCl, 250 mM Imidazole, pH 8 
Desalting buffer 50 mM KPi, 300 mM NaCl, 10% (w/v) glycerol, pH 7.5 
Table S3: Buffer Composition 
 
8 
 
Enzyme Immobilization (Batch Mode) 
In batch mode, 10 mg of cryogel was weighed and added to a 1 mL solution of 50 mM potassium 
phosphate buffer (KPB) (300 mM de NaCl, 10 %  glycerol pH 7.5) containing 1 mg of B9L0N2 enzyme, 
resulting in a 10% enzyme loading on the cryogel. The suspension was stirred at 800 rpm for 2 hours 
at 4°C to facilitate enzyme immobilization. Samples were taken at intervals (10, 20, 30, 60, 90, and 120 
minutes), followed by centrifugation at 4000–6000 rpm for 2 to 5 minutes to separate the solid cryogel 
from the supernatant. After the final sampling, the supernatant was discarded, and the solid cryogel 
was washed twice with the buffer solution to remove unbound enzyme. Each wash involved stirring 
the suspension for 3 minutes at 4°C, followed by centrifugation. 
Enzyme Quantification by Bradford Assay 
The amount of enzyme immobilized on the cryogel was determined using the Bradford protein assay. 
A standard curve was prepared using BSA (0.1 g/L) in the same 50 mM KPB (300 mM de NaCl, 10 %  
glycerol pH 7.5). Bradford reagent was added to each sample, including the immobilized enzyme 
supernatant samples, and incubated for 15 minutes in the dark. The absorbance was measured at 595 
nm. The protein content in the samples was calculated by comparison with the standard curve, and 
the amount of enzyme immobilized was determined based on the amount of protein remaining in the 
supernatant after immobilization. 
Enzyme Activity of Immobilized B9L0N2 
The catalytic activity of immobilized B9L0N2 was measured by following the transamination of 
pyruvate in presence of racemic α-methylbenzylamine to alanine and acetophenone. Transaminase 
uses the cofactor pyridoxal phosphate (PLP) to carry out this reaction. The reaction mixture contained 
25 mM pyruvate, 25 mM racemic α-methylbenzylamine, 0.1 mM PLP, and 1% DMSO in 50 mM KPB 
(300 mM NaCl, pH 7.5). The initial enzyme/substrate ratio is fixed at 0.025. The reaction was initiated 
by adding 10 mg of enzyme-loaded cryogel to the mixture and stirred at 25°C for 10 minutes at 800 
rpm. Acetophenone formation was monitored at 245 nm using UV spectrophotometry. The activity of 
the immobilized enzyme was evaluated over five reaction cycles, with each cycle involving the same 
process followed by washing the cryogel and re-initiating the next reaction. 
Continuous Use of B9L0N2 Enzyme 
In continuous mode (see figure S2 for continuous flow set-up), 70 mg of cryogel was placed in a 
thermostatted glass column maintained at 4°C using a cryostat. The enzyme solution, prepared in 
50mM KPB (300 mM NaCl, 10 %  glycerol, pH 7.5), was percolated through the column at a flow rate 
of 0.1 mL/min using an HPLC pump in order to result in a 5% enzyme loading. Following enzyme 
immobilization, the column was flushed with 5 mL of Tris buffer at the same flow rate to remove 
unbound proteins. The continuous enzymatic reaction was performed by circulating a reaction mixture 
containing 25 mM pyruvate, 25 mM racemic α-methylbenzylamine, 0.1 mM PLP in 50 mM KPB (300 
mM NaCl, 1% DMSO, pH 7.5) through the column. In circulation mode, 10 mL of the reaction mixture 
was passed through the column at 0.1 mL/min for up to 5 hours, with 100 µL samples taken hourly. 
The acetophenone formation was monitored at 245 nm to determine conversion ratio. 
 


Innovative carrier materials for advancing enzyme immobilization in industrial biocatalysis

Ahlem Rahali

Norman Le Floch

Fatma Allouche

Henri Doucet

Synthesis of C5-arylated pyrazoles from a pyrazole-4-carboxylate by a palladium-catalyzed 
C5–H bond activation/C4-decarboxylation sequence.  
Ahlem Rahali,a,b Norman Le Floch,a Fatma Allouche,b,* and Henri Douceta,* 
a Univ Rennes, CNRS, ISCR-UMR 6226, F-35000 Rennes, France. 
b Laboratoire de Chimie Médicinale et Environnementale, Institut Supérieur de Biotechnologie, 
Route de la Soukra km 4, 3038 Sfax, Tunisia. 
Supplementary material 
Copy of 1H, 13C and 19F NMR spectra of all products S2-S33 
Ethyl 1-methyl-5-(4-(trifluoromethyl)phenyl)pyrazole-4-carboxylate (1) 
 
 
 
  
Ethyl 5-(4-cyanophenyl)-1-methylpyrazole-4-carboxylate (2) 
 
 
  
Ethyl 5-(4-acetylphenyl)-1-methylpyrazole-4-carboxylate (3) 
 
 
  
Ethyl 1-methyl-5-(4-propionylphenyl)pyrazole-4-carboxylate (4) 
 
 
  
Ethyl 5-(4-formylphenyl)-1-methylpyrazole-4-carboxylate (5) 
 
 
  
Ethyl 5-(4-(ethoxycarbonyl)phenyl)-1-methylpyrazole-4-carboxylate (6) 
 
  
Ethyl 1-methyl-5-(4-(trifluoromethoxy)phenyl)pyrazole-4-carboxylate (7) 
 
 
 
  
Ethyl 5-(4-(difluoromethoxy)phenyl)-1-methylpyrazole-4-carboxylate (8) 
 
 
 
  
Ethyl 5-(4-fluorophenyl)-1-methylpyrazole-4-carboxylate (9) 
 
 
 
  
Ethyl 5-(4-chlorophenyl)-1-methylpyrazole-4-carboxylate (10) 
 
 
  
Ethyl 5-(4-(tert-butyl)phenyl)-1-methylpyrazole-4-carboxylate (11) 
 
  
Ethyl 5-(3-cyanophenyl)-1-methylpyrazole-4-carboxylate (12) 
 
  
Ethyl 5-(3-formylphenyl)-1-methylpyrazole-4-carboxylate (13) 
 
  
Ethyl 1-methyl-5-(3-(trifluoromethyl)phenyl)pyrazole-4-carboxylate (14) 
 
 
 
  
Ethyl 5-(3-fluorophenyl)-1-methylpyrazole-4-carboxylate (15) 
 
 
 
  
Ethyl 5-(3-chlorophenyl)-1-methylpyrazole-4-carboxylate (16) 
 
  
Ethyl 5-(3-methoxyphenyl)-1-methylpyrazole-4-carboxylate (17) 
 
  
Ethyl 5-(2-cyanophenyl)-1-methylpyrazole-4-carboxylate (18) 
 
 
  
Ethyl 5-(3,5-bis(trifluoromethyl)phenyl)-1-methylpyrazole-4-carboxylate (19) 
 
 
 
  
Ethyl 5-(3,5-difluorophenyl)-1-methylpyrazole-4-carboxylate (20) 
 
 
  
Ethyl 1-methyl-5-(pyridin-3-yl)pyrazole-4-carboxylate (21) 
 
 
  
Ethyl 1-methyl-5-(pyridin-4-yl)pyrazole-4-carboxylate (22) 
 
 
  
1-Methyl-5-(4-(trifluoromethyl)phenyl)pyrazole (23) 
 
 
 


Synthesis of C5-arylated pyrazoles from a pyrazole-4-carboxylate by a palladium-catalyzed C5–H bond activation/C4-decarboxylation sequence

Keyu Mao

Kristina Plevova

Xi Chen

Laura Juliana Prieto Pabon

Sophie  Poulain-Martini

Véronique Michelet

Silver-, gold-, and platinum-catalyzed [4+2] cyclizations: synthesis of 2,3,3a,4-tetrahydro-1H-benzo[f]isoindol-1-one derivatives

Javid Rzayev

Zhenbang Chang

Tatiana Besset

Toolbox for emerging fluorinated groups

Lucie Imbernon

Myriam Pire

Evdokia K. Oikonomou

Szilvia Karpati

Sylvie Tencé-Girault

Sophie Norvez

Highlights of epoxidized natural rubber: from self-vulcanizing blends to reprocessable chemical or physical networks

Not a member yet

Lectins in the sweet strategy of lung pathogens: from structural glycobiology to design of glycomimetic pathoblockers

Molecular aspects of cell-penetrating peptides: key amino acids, membrane partners, and non-covalent interactions

Locally activated semisynthetic fluorescent biosensors for imaging cellular biochemistry

In search of a viable route to high-melting olefinic block copolymers

Extending the toolbox for enzymatic carboligation: synthesis of α-hydroxyketones catalyzed by thermostable transketolase from <i>Geobacillus stearothermophilus</i>

Innovative carrier materials for advancing enzyme immobilization in industrial biocatalysis

Synthesis of C5-arylated pyrazoles from a pyrazole-4-carboxylate by a palladium-catalyzed C5–H bond activation/C4-decarboxylation sequence

Silver-, gold-, and platinum-catalyzed [4+2] cyclizations: synthesis of 2,3,3<i>a</i>,4-tetrahydro-1<i>H</i>-benzo[<i>f</i>]isoindol-1-one derivatives

Toolbox for emerging fluorinated groups

Highlights of epoxidized natural rubber: from self-vulcanizing blends to reprocessable chemical or physical networks

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